Chemistry, toxicology & urinalysis

• Hepatitis B testing is used for screening, diagnosis and monitoring of hepatitis B virus (HBV) infections, which involves measurement of HBV specific antigens, antibodies and HBV DNA
• Test results of individual HBV markers or a combination of markers can help determine whether a patient has acute or chronic HBV infection, is immune to HBV due to past infection or vaccination or is susceptible to HBV infection as well as provide information on the level of viral replication and infectivity

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• Overview
  • HBV is transmitted through contact with infected blood or body fluids, such as during pregnancy or delivery, through sex or by injection drug use, with the greatest risk for chronic infection occurring during perinatal infection
  • Coinfection with other viruses is seen with HBV infection, such as hepatitis D virus, hepatitis C virus and human immunodeficiency virus (HIV)
  • Clinical manifestation of acute HBV infection varies from asymptomatic (~70%) to symptomatic (~30%) hepatitis presenting with nonspecific symptoms (i.e., fatigue, anorexia, nausea, right upper quadrant discomfort and jaundice) and uncommonly, fulminant hepatitis (encephalopathy and coagulopathy, < 1% in immunocompetent adults) (Lancet 2023;401:1039)
  • Acute viral hepatitis infections are usually associated with significant elevation of aminotransferases (10 - 100 times the upper limit of normal range), such as alanine transaminase (ALT) and aspartate transaminase (AST), with AST/ALT ratio typically < 1 (Clarke: Contemporary Practice in Clinical Chemistry, 4th Edition, 2020)
  • Patients with chronic HBV infection have an increased risk for liver related complications, including cirrhosis, liver failure and HCC
• Acute HBV infection
  • Incubation period of HBV can range from 1 to 6 months; HBV DNA may be the only marker detectable in the first 2 weeks, whereas HBsAg appears in the serum 2 - 10 weeks after exposure, before symptom onset and elevated aminotransferases (see Figure 1)
  • In the early phase of an acute infection, HBsAg, IgM anti-HBc and HBeAg are all positive with HBV DNA detectable in high levels
  • During recovery, HBsAg disappears and seroconverts to anti-HBs
  • In resolved hepatitis B, total anti-HBc will remain positive and anti-HBs will develop to protect against future infection; HBV DNA is undetectable in serum but can persist in the liver
• Chronic HBV infection
  • Chronic hepatitis B is defined as an HBV infection lasting > 6 months, typically with HBsAg and total anti-HBc present but not IgM anti-HBc (Hepatology 2018;67:1560)
    • HBeAg and anti-HBe are variably present and HBV DNA levels also vary (see Figure 1)
  • Patients with chronic HBV infection may go through 5 different clinical phases, though not all patients go through all phases: immune tolerant, immune active, inactive carrier, HBeAg negative immune active and resolved or occult HBV infection
  • Some patients with chronic HBV infection eventually become HBsAg negative
• Isolated total anti-HBc positive is used to describe the serological pattern with positive total anti-HBc but negative HBsAg and anti-HBs
• Occult hepatitis B refers to a small fraction of isolated total anti-HBc positive individuals who present with low level chronic viremia, in whom HBsAg is absent in the serum but HBV DNA is detectable

Clinical phases of chronic HBV infection (adapted from Lancet 2023;401:1039)

Clinical phases	Alanine transaminase	HBeAg	Anti-HBe	HBsAg	Anti-HBs	HBV DNA
Immune tolerant	Normal	+	-	+	-	++++
Immune active	Elevated	+	-	+	-	++++
Inactive carrier	Normal	-	+	+	-	++
HBeAg negative Immune active	Elevated	-	+ / -	+	-	++
Resolved or occult HBV infection	Normal	-	+	-	+ / -	+ / -

• Methodology
  • Chemiluminescent immunoassay (CLIA) is the most common platform used for hepatitis B serology testing
• HBsAg
  • Protein on the surface of HBV that can be detected in either acute or chronic infections
  • Presence of HBsAg indicates infection, with exception of transient positivity within 30 days after a dose of hepatitis B vaccine (CDC: Viral Hepatitis - Interpretation of Hepatitis B Serologic Test Results [Accessed 19 February 2024])
  • Positive HBsAg specimens are required to be retested by a neutralization assay, which uses the principle of specific antibody neutralization to confirm the presence of HBsAg
  • Some HBsAg mutants are not detectable by older assays
    • HBsAg mutant strains can be detected by some HBsAg assays that first became available in the United States in 2015, including Abbott ARCHITECT instrument, ETI-MAK-2 PLUS and Siemens ADVIA Centaur XP or XPT instrument (Kidney Med 2019;1:347)
    • Alternative testing options include quantitative HBV DNA or total anti-HBc with follow up HBV DNA if positive
• Anti-HBs
  • Antibodies develop as part of the normal immune response to HBV infection or hepatitis B vaccination
  • Appearance of anti-HBs and decline of HBsAg indicates recovery from HBV infection and development of immunity
  • Anti-HBs concentration > 10 mIU/mL after completion of a vaccine series indicates immunity
  • Testing for anti-HBs within 6 months of hepatitis B immune globulin administration can lead to inaccurate assessment of immune status (Dev Biol Stand 1983;54:347)
• Total anti-HBc
  • Antibodies develop in all HBV infections (resolved or current) and typically persist for life
  • Immunity gained through vaccination does not develop anti-HBc
  • Total anti-HBc detects both IgG and IgM antibodies to HBcAg
  • No commercially available test for IgG anti-HBc alone
• IgM anti-HBc
  • Antibodies develop after HBsAg during the early phase of HBV infection and typically wane around 1 year postexposure in both acute and chronic HBV infections
  • During acute HBV infection, there can be a period of 1 - 6 months between the loss of HBsAg and the appearance of anti-HBs, when diagnosis has to rely on IgM anti-HBc
  • IgM anti-HBc should only be tested when acute HBV infection is a concern
• Other HBV markers
  • HBeAg is a marker for viral replication and high infectivity; HBeAg is positive during acute HBV infection and remains positive in the early phases of chronic HBV infection
  • Anti-HBe can be used to monitor response to treatment and progression of chronic HBV infection; positive anti-HBe with negative HBeAg are seen in patients with resolved HBV infection or in the later phases of chronic HBV infection
  • HBV DNA test is a measure of viral load, which indicates HBV replication status to guide treatment decisions and to assess response; HBV DNA levels can also predict the risk of cirrhosis and HCC
  • Emerging tests may provide additional information on patients with chronic HBV infection, such as quantification of HBsAg, HBV RNA and hepatitis B core related antigen (HBcrAg); HBV RNA and HBcrAg are considered more reliable serum markers of cccDNA transcription than HBV DNA or HBsAg level, which can be derived from both cccDNA and integrated HBV DNA (Gastroenterology 2019;156:355)

Hepatitis B testing results interpretation (adapted from MMWR Recomm Rep 2018;67:1)

HBsAg	Total anti- HBc	IgM anti- HBc	Anti- HBs	HBV DNA	Possible interpretation
-	-	-	-	-	Never infected; susceptible if never vaccinated or vaccine failure
+	-	-	-	+ or -	Early acute infection (if HBV DNA is positive); transiently positive for HBsAg after vaccination (if HBV DNA is negative)
+	+	+	-	+	Acute infection
-	+	+	+ or -	+ or -	Acute resolving infection: window period if anti-HBs is negative
-	+	-	+	-	Recovered from past infection and immune
+	+	-	-	+	Chronic HBV infection
-	-	-	+	-	Immune from vaccination; passive anti-HBs transfer after hepatitis B immune globulin administration
-	+	-	-	+ or -	Isolated total anti-HBc positive*
-	+ or -	-	+ or -	+	Occult HBV infection
+ or -	+	+ or -	+ or -	+	Possible HBsAg mutant infection

• *Isolated total anti-HBc positive could result from
  • Loss of anti-HBs after past resolved infection; HBV DNA is negative
  • False positive total anti-HBc (i.e., susceptible); HBV DNA is negative
    • To resolve the ambiguity of a false positive total anti-HBc result, test a follow up sample 4 - 8 weeks later
    • If found positive, interpret as a resolved infection
    • If negative, interpret as false positive
  • Passive maternal transfer of total anti-HBc to infant born to a HBsAg positive gestational parent for up to 24 months; HBV DNA is negative
  • Occult HBV infection: HBV DNA is positive, typically at low levels; anti-HBs may be positive or negative
  • HBsAg mutant infection: HBV DNA is positive, typically at high levels; anti-HBs may be positive or negative

Which of the following is the most appropriate test for the diagnosis of acute hepatitis B virus (HBV) infection?

1. Hepatitis B DNA testing
2. Hepatitis B e antigen (HBeAg)
3. Hepatitis B surface antibody (anti-HBsAg)
4. Hepatitis B surface antigen (HBsAg)
5. Total antibody to hepatitis B core antigen (total anti-HBc)

D. Hepatitis B surface antigen (HBsAg). The presence of HBsAg indicates HBV infection, either acute or chronic. Answers A, B, C and E are incorrect because these tests are useful for differentiating infection phases and assessing infectivity or immunity but cannot be used for the diagnosis of acute HBV infection.

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Reference: Hepatitis B testing

HBsAg	Negative
Total anti-HBc	Positive
IgM anti-HBc	Positive
Anti-HBsAg	Negative
HBeAg	Negative
HBV DNA	Negative

What is the possible interpretation of the hepatitis B testing results shown above?

1. Active chronic HBV infection
2. Early acute HBV infection
3. Occult HBV infection
4. Recovered from past HBV infection and immune
5. Resolving acute HBV infection (window period)

E. Resolving acute infection (window period). During acute HBV infection, there can be a period of 1 - 6 months between the loss of HBsAg and the appearance of anti-HBs, when diagnosis has to rely on IgM anti-HBc. Answer B is incorrect because HBsAg, HBeAg and HBV DNA are negative. Answer D is incorrect because anti-HBsAg is negative, indicating a lack of immunity. Answers A and C are incorrect because IgM anti-HBc is positive.

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Reference: Hepatitis B testing